人肾近端小管上皮细胞的原代培养、传代及鉴定方法研究

Primary Culture, Subculture and Identification of Human Renal Proximal Tubule Cells

目的:创建一种重复性好、细胞量多的人肾近端小管细胞(PTC)培养方法.方法:取新鲜正常人肾组织分离纯化,用含10%新生牛血清等营养充分的DMEM/F12(1:1)液进行近端小管细胞的原代培养及传代,并以免疫组化、酶化学染色、透射电镜鉴定.结果:培养5～6天后融合成单层细胞,形态呈鹅卵石样,可传代7～9代,鉴定确定为人肾近端小管细胞,重复培养10次,均能稳定获得同样细胞,每克肾皮质可分离培养出(6～12)×106个PTC.结论:此培养方法可在7天内获得人肾近端小管细胞,且重复性好,细胞数量多,为进行肾小管细胞治疗提供必需的细胞,也为研究肾小管病变提供实验平台.

Objective: To establish a duplicable and efficient culture method of human proximal tubule cells(PTC) in vitro. Methods: Separated and purified human PTC were cultured in DMEM/F12(1∶1) supplemented with 10% fetal bovine serum. The bred cells were subcultured and identitied by immunocytochemical and histochemical enzyme staining, transmission electron microscopy. Results: After 5-6 days, the cultured cells reached confluence,displaying the typical cobblestone appearance and could be subcultured till 7-9 passages. They were validated as human PTC by method of cell morphology and staining of specific marker. This method had replicated for ten times and amplified the same cells. Conclusion:This culture system can amplify a lot of human PTC in 7 days .These bred cells can supply for cell therapy and serve as a material for advanced study .